We are studying mechanisms of beta-lactam antibiotic resistance. We are using new methodologies in the area combining quantum mechanics and molecular mechanics calculations, free energy perturbations, and continuum electrostatics models to examine the hydrolytic activity against beta- lactam antibiotics displayed by the bacterial defensive enzymes which render bacterial resistance--TEM-1 from E. coli and E. cloacae P99 beta- lactamases which are common in clinical pathogens. We are investigating the mechanism of catalysis on simple models of methanol-mediated hydrolysis of small beta-lactam molecules in the gas phase and in the solution followed by the modeling for the methanol-mediated hydrolysis of benzylpenicillin--a typical beta-lactam antibiotic. We have just completed a part of the project on calculation titration curves for the representative beta-lactamases. The results of this work helped to elucidate mechanisms of activation of the critical active-site residues in these enzymes. This data will be used in combination with the study of the entire mechanism of the beta-lactam antibiotic hydrolysis by beta-lactamases on a higher level of theory. The detailed knowledge gained from the science disclosed in this research project should ultimately be a driving force in designing novel potent antimicrobials that overcome the problem of bacterial resistance.